Organisms transcribe genetic information stored in the genome to mRNA (gene expression) and synthesize proteins on the basis of the information. Furthermore, the living organisms do life activities by the biological functions of the proteins. In recent years, studies for comprehensive understanding of the living organisms have advanced rapidly by exhaustively analysis of biological functions at a molecular level. By the exhaustive analysis, for example, the functions of pathological cells or immunocytes can be elucidated and applied to the understanding of the causes of diseases or the development of new drugs.
Genome analysis capable of more directly obtaining information on the biological functions, or gene expression analysis based on mRNA expression levels has received attention as a means of conducting the exhaustive analysis. The gene expression analysis mainly has been conducted by using DNA microarray. But in recent years, this analysis is mainly conducted by using a large-capacity DNA sequencer. Usually, cultured cells or tissues, which composed of a large number of cells, are used as analysis samples. However, a gene expression profiles even within homogeneous tissue differs among individual cells or times and is therefore not always the same. Thus, for understanding the functions of a tissue accurately and in detail, it is required to analyze gene expression in each individual cell constituting the tissue and comprehensively grasp the whole tissue on the basis of the information on the gene expression. Nonetheless, an mRNA level derived from a single cell is very small. Therefore, it has heretofore been difficult to conduct the gene expression analysis of each individual cell in a tissue. However, with technical progress in nucleic acid analysis apparatuses, reagents, etc., the analysis of a genome sequence, an mRNA sequence, or a gene expression profiles derived from a single cell has become feasible in recent years (Non Patent Literature 1). Particularly, methods for analyzing gene expression in a single cell using a large-capacity next-generation DNA sequencer have achieved remarkable development. Now, an expression status can be determined in detail as to an enormous number of genes (Non Patent Literatures 2 and 3).
In an organism composed of a large number of cells, these cells do not work each independently, but are involved in each other through the mutual exchange of information. Therefore, for knowing life phenomena in detail, it is required to not only analyze gene expression analysis in a single cell but analyze many cells present in the neighborhood of the cell at the same time and one by one. The number of cells constituting the cell group to be analyzed is several hundreds or in some cases, beyond several tens of thousands. In the case of conducting the gene expression analysis of each individual cell in the cell group, reaction must be carried out in separate reaction vessels by each cell. However, the number of cells that can be analyzed at once relatively easily by a current nucleic acid analysis technique is on the order of several tens of cells. Hence, there has been a demand for a novel technique for extracting nucleic acids such as mRNA on a cell basis from several hundreds or more cells and analyzing the nucleic acids at once.
Patent Literature 1 discloses a method for constructing a complementary-strand DNA (cDNA) library using a porous membrane, etc., as a means for solving the problems described above. In this method, a large number of cells or living tissue sections are located on a membrane, and mRNA can be extracted therefrom under an electric field to construct cDNA library arrays on a cell basis in a multiple regions present immediately beneath the respective cells. In the cDNA library arrays constructed by this method, the cDNA library derived from a single cell is located in each region in a two-dimensional planar form. For analyzing the cDNA library arrays containing multiple regions at once by using a large-capacity DNA sequencer, nucleic acid amplification step and fragmentation step of cleaving DNA into a length convenient for nucleotide sequence analysis are necessary. During the course of these steps, however, reaction products are mixed up among the regions. Therefore, the identity of a cell from the resulting nucleotide sequence information is masked.